Triglycerides contribute to the increased cardiovascular mortality in people with diabetes. Long-chain fatty acids are released from triglycerides in lesions of atherosclerosis, and are esterified to CoA in order to be utilized by vascular cells through a reaction that is catalyzed by long-chain acyI-CoA synthetases (ACS1-5). Oleic acid (OA), the most common fatty acid in triglycerides, has important pro-atherosclerotic effects in vascular cells. Four questions will be addressed: 1. Which ACS isoforms are expressed in primary smooth muscle cells (SMCs) and macrophages, and are they regulated by glucose and lipids? We hypothesize that primary SMCs and macrophages express several ACS isoforms, and that their expression is differentially regulated by glucose and lipids in vitro and in vivo. For the in vivo studies, we have developed a new transgenic mouse model of diabetes-accelerated atherosclerosis. 2. Is ACS2 necessary for OA incorporation into phosphatidylcholine and generation of OA-enriched 1,2-diacylglycerol following growth factor stimulation in SMCs? OA enhances the mitogenic effects of growth factors in SMCs by generation of OA-enriched 1,2-diacylglycerol (1,2-DAG) following growth factor-stimulation of phospholipase D. We hypothesize that ACS2 mediates OA-incorporation into phosphatidylcholine and generation of OA-enriched 1,2-DAG following growth factor stimulation. We propose to inhibit ACS2 by using ACS inhibitors and RNAi. 3. Is ACS2 necessary for the ability of OA to enhance growth factor-induced proliferation in SMCs? We hypothesize that ACS2 is necessary for the ability of OA to enhance growth factor-induced proliferation in SMCs. Inhibitors of ACS isoforms and RNAi will be used to inhibit OA-mediated potentiation of mitogenic effects of growth factors. 4. Which specific ACS isoform(s) is necessary for OA-induced macrophage death and inhibition of cholesterol efflux? OA induces macrophage death and inhibits cholesterol efflux. We hypothesize that a specific ACS isoform is necessary for these events. ACS inhibitors and RNAi will be used to determine the ACS isoform that mediates OA-induced effects on macrophage death, cholesterol efflux and lipid metabolism. The ACS isoforms in vascular cells have not been studied to date. We expect to identify ACS isoforms required for important biological effects of OA in primary smooth muscle and macrophages. ? ?
